Method for optical resolution of racemic 3-hydroxybutyric acid

Abstract

To provide methods for optical resolution of racemic 3-hydroxybutyric acid or salts thereof to enable production of optically active 3-hydroxybutyric acid.SOLUTION: Disclosed is a method of optical resolution of racemic 3-hydroxybutyric acid or salt thereof, comprising the steps of: aerobically culturing a halophilic bacterium of the genus Halomonas in a culture medium containing an inorganic salt and racemic 3-hydroxybutyric acid or a salt thereof as an organic carbon source; separating the cells from the culture fluid after the aerobic culture; and subjecting the separated cells to microaerobic culture in an inorganic salt-containing culture medium to produce (R)-3-hydroxybutyric acid or salt thereof in the culture fluid.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for optical resolution of racemic 3-hydroxybutyric acid. [Background technology]

[0002] 3-Hydroxybutyric acid and its salts (hereinafter simply referred to as "3HB") are naturally occurring substances in the human body, making them highly biocompatible and promising as an innovative energy source to replace carbohydrates. Furthermore, 3HB has been shown to function not only as an energy source but also as a signaling molecule that affects the expression of various genes and protein activity. For example, 3HB is known to improve cognitive function and long-term memory by inhibiting histone deacetylase through its gene expression regulation effect, and has been confirmed to be effective in preventing Alzheimer's disease. Furthermore, 3HB, which is produced through the ingestion and metabolism of medium-chain fatty acids, which are abundant in coconut oil, has been shown to improve the symptoms of patients with Alzheimer's disease and diabetes, who have difficulty effectively utilizing carbohydrates in the brain and body. Furthermore, because 3HB is converted into energy more quickly than carbohydrates in the body and has the effect of suppressing the absorption of fat and sugar into cells, it is actively used as an energy source for athletes and in diet and health foods.

[0003] In addition to these uses, 3HB is known to be useful as a raw material for biodegradable resins, and its value in industrial applications is also increasing.

[0004] Since optical activity is often advantageous for the use of compounds, not limited to 3HB, in various applications, there is a demand for a technique for separating optically active compounds by optical resolution of a racemate. For example, a method for producing optically active hydroxycarboxylic acid esters or optically active hydroxycarboxylic acids by optical resolution of racemic hydroxycarboxylic acid esters has been proposed (Patent Document 1).

[0005] In the production method described in Patent Document 1, a racemic hydroxycarboxylic acid ester is subjected to the action of a microorganism belonging to the genus Enterobacter or Chitrobacter and having carboxylic acid ester decomposition activity, or a bacterial cell obtained from a culture medium of the microorganism, etc. In this way, the carboxylic acid ester is stereoselectively hydrolyzed and optically resolved, and one of the remaining optically active hydroxycarboxylic acid esters or the other optically active hydroxycarboxylic acid obtained by hydrolysis is collected. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 4069742 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the production method described in Patent Document 1 is a technology limited to racemic hydroxycarboxylic acid esters, and therefore cannot produce optically active 3-hydroxybutyric acid or a salt thereof by optical resolution of racemic 3-hydroxybutyric acid or a salt thereof.

[0008] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for optically resolving racemic 3-hydroxybutyric acid or a salt thereof, which enables the production of optically active 3-hydroxybutyric acid. [Means for solving the problem]

[0009] The method for optical resolution of racemic 3-hydroxybutyric acid according to the present invention for achieving the above object is characterized by the following features: A method for optical resolution of racemic 3-hydroxybutyric acid or a salt thereof, comprising the steps of: an aerobic culturing step of aerobically culturing a halophilic bacterium belonging to the genus Halomonas in a medium containing inorganic salts and racemic 3-hydroxybutyric acid or a salt thereof as an organic carbon source; a separation step of separating bacterial cells from the culture solution after the aerobic culture step; The bacterial cells separated in the separation step are microaerophilically cultured in a medium containing inorganic salts, and a microaerophilic culture step is carried out in this order to produce (R)-3-hydroxybutyric acid or a salt thereof in the culture solution.

[0010] As a result of extensive research, the present inventors have discovered that when halophilic bacteria belonging to the genus Halomonas are aerobically cultured in a medium containing racemic 3-hydroxybutyric acid or a salt thereof as an organic carbon source, the halophilic bacteria preferentially take up (R)-3-hydroxybutyric acid and accumulate it as poly[(R)-3-hydroxybutyric acid] (hereinafter also referred to as "PHB"), thereby completing the present invention.

[0011] That is, according to the above-mentioned characteristic configuration, in the aerobic culture step, the halophilic bacterium preferentially takes up (R)-3-hydroxybutyric acid from racemic 3-hydroxybutyric acid, which accumulates as PHB within the bacterial cells, while most of the (S)-3-hydroxybutyric acid remains in the culture solution. Therefore, (S)-3-hydroxybutyric acid is present in the culture solution from which the bacterial cells were separated in the separation step. Then, by microaerophilically culturing the bacterial cells obtained in the separation step, (R)-3-hydroxybutyric acid is secreted and produced outside the bacterial cells (into the culture solution). Thus, according to the method for optical resolution of racemic 3-hydroxybutyric acid having the above-mentioned characteristic configuration, racemic 3-hydroxybutyric acid can be separated into (R)-3-hydroxybutyric acid and (S)-3-hydroxybutyric acid.

[0012] A further characteristic feature of the method for optical resolution of racemic 3-hydroxybutyric acid according to the present invention is: The feature is that an R-isomer recovery step is carried out to recover (R)-3-hydroxybutyric acid or a salt thereof from the culture solution obtained in the microaerobic culture step.

[0013] As described above, the culture solution obtained in the microaerobic culture step contains (R)-3-hydroxybutyric acid or a salt thereof secreted and produced by the bacterial cells. According to the above-described characteristic configuration, (R)-3-hydroxybutyric acid or a salt thereof can be recovered from the culture solution by the R-isomer recovery step. Therefore, the optical resolution method having the above-described characteristic configuration makes it possible to produce (R)-3-hydroxybutyric acid as optically active 3-hydroxybutyric acid from racemic 3-hydroxybutyric acid.

[0014] A further characteristic feature of the method for optical resolution of racemic 3-hydroxybutyric acid according to the present invention is: The difference is that an S-isomer recovery step is carried out to recover (S)-3-hydroxybutyric acid or a salt thereof from the culture medium from which the bacterial cells have been separated in the separation step.

[0015] As described above, the culture medium from which the bacterial cells have been separated in the separation step contains (S)-3-hydroxybutyric acid, which is not easily taken up by halophilic bacteria. According to the above-mentioned characteristic configuration, (S)-3-hydroxybutyric acid or a salt thereof can be recovered from the culture medium from which the bacterial cells have been separated by the S-isomer recovery step. Therefore, the method for optical resolution of racemic 3-hydroxybutyric acid having the above-mentioned characteristic configuration makes it possible to produce (S)-3-hydroxybutyric acid as optically active 3-hydroxybutyric acid from racemic 3-hydroxybutyric acid.

[0016] A further characteristic feature of the method for optical resolution of racemic 3-hydroxybutyric acid according to the present invention is: The halophilic bacterium is Halomonas sp. KM-1 strain.

[0017] The inventors of the present application have experimentally confirmed that when the halophilic bacterium is Halomonas sp. KM-1 strain, the Halomonas sp. KM-1 strain preferentially takes up (R)-3-hydroxybutyrate from racemic 3-hydroxybutyrate, accumulates PHB within the bacterial cells, and secretes and produces (R)-3-hydroxybutyrate into the culture medium by switching to microaerobic culture. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a diagram for explaining an outline of a method for optical resolution of racemic 3-hydroxybutyric acid according to an example. [Figure 2] 1 is a graph showing analytical data during aerobic culture. [Figure 3] 1 is a graph showing analytical data during microaerobic culture. DETAILED DESCRIPTION OF THE INVENTION

[0019] The method for optical resolution of racemic 3-hydroxybutyric acid according to the present invention will be described below. Preferred examples are described below, but these examples are provided to more specifically illustrate the present invention, and various modifications are possible within the scope of the present invention, and the present invention is not limited to the following descriptions.

[0020] [Outline of optical resolution method for racemic 3-hydroxybutyric acid] The method for optical resolution of racemic 3-hydroxybutyric acid according to an embodiment of the present invention comprises the following steps. (1) A step of aerobically culturing a halophilic bacterium belonging to the genus Halomonas in a medium containing inorganic salts and racemic 3-hydroxybutyric acid or a salt thereof as an organic carbon source. (2) A separation step of separating the bacterial cells from the culture solution after the aerobic culture step. (3) A microaerophilic culturing step in which the bacterial cells separated in the separation step are microaerophilically cultured in a medium containing inorganic salts to produce (R)-3-hydroxybutyric acid or a salt thereof in the culture solution.

[0021] Furthermore, in this embodiment, the following steps are further carried out. (4) An R-isomer recovery step for recovering (R)-3-hydroxybutyric acid or a salt thereof from the culture medium obtained in the microaerobic culture step. (5) An S-isomer recovery step for recovering (S)-3-hydroxybutyric acid or a salt thereof from the culture medium from which the bacterial cells have been separated in the separation step.

[0022] [Aerobic culture process] In the aerobic culture step of the optical splitting method of the present invention, an aerobic culture of a halophilic bacterium belonging to the genus Halomonas is performed in a medium. <A: Halophilic bacterium belonging to the genus Halomonas> The halophilic bacterium belonging to the genus Halomonas used in the present invention preferably grows aerobically in a medium containing inorganic salts and racemic 3-hydroxybutyric acid (racemic 3HB), takes up (R)-3-hydroxybutyric acid (R-3HB) into the cells, accumulates it as poly[(R)-3-hydroxybutyric acid] (PHB), and can secrete and produce R-3HB or its salt into the extracellular medium by changing the culture conditions. Any bacterium with such properties may be used.

[0023] The halophilic bacterium belonging to the genus Halomonas has a halophilic property suitable for a salt concentration of 0.1 to 1.0 M and can sometimes grow even in a medium without salt. And the above-mentioned halophilic bacterium belonging to the genus Halomonas usually grows in a medium with a pH of about 5 to 12.

[0024] Examples of the halophilic bacterium belonging to the genus Halomonas include Halomonas sp. KM-1 strain. The Halomonas sp. KM-1 strain was deposited on July 10, 2007, at the Patent Biological Depositary, National Institute of Advanced Industrial Science and Technology (1-1-1 Higashi, Tsukuba, Ibaraki 305-8566) under the accession number FERM P-21316. Further, this strain has now been transferred to an international deposit, and its accession number is FERM BP-10995. The 16S rRNA gene of the Halomonas sp. KM-1 strain is registered in DDBJ under Accession Number AB477015.

[0025] Also, in view of the growth characteristics of the above-mentioned halophilic bacteria, etc., the halophilic bacteria used in the aerobic culture step in the present invention are those that preferentially take up R-3HB into the cells, accumulate PHB, and then secrete R-3HB in the microaerobic culture step described later, and are not limited to the Halomonas sp. KM-1 strain. Examples of such halophilic bacteria belonging to the genus Halomonas include Halomonas pantelleriensis (ATCC 700273) and Halomonas campisalis (ATCC 7000597).

[0026] Furthermore, from the analysis by 16S ribosomal RNA sequence, not only the above-mentioned halophilic bacteria, but also Halomonas nitritophilus, Halomonas alimentaria, etc. may be used as halophilic bacteria belonging to the genus Halomonas used in the aerobic culture step.

[0027] In addition, genes may be introduced into the above-mentioned halophilic bacteria belonging to the genus Halomonas. The gene to be introduced is not particularly limited as long as it does not affect the property of preferentially taking up R-3HB among the racemic 3HB, accumulating PHB, and then secreting R-3HB in the microaerobic culture step described later in the optical resolution method according to the present invention. As the method for introducing the recombinant DNA into the cells and the transformation method thereby, various general methods can be adopted.

[0028] <B: Medium> The medium used in the aerobic culture step contains inorganic salts and racemic 3HB or its salt as an organic carbon source. The pH of the medium is not particularly limited as long as it satisfies the growth conditions of the above-mentioned halophilic bacteria, but specifically, it may be about pH 5 to 12, and more preferably pH 8.8 to 12. In addition, using an alkaline medium is preferable because it can effectively prevent contamination by other bacteria.

[0029] Also, the medium may be a liquid medium or a solid medium.

[0030] The inorganic salts to be added to the medium used in the aerobic culture step are not particularly limited, and examples thereof include phosphates, nitrates, carbonates, sulfates, and metal salts such as sodium, magnesium, potassium, manganese, iron, zinc, copper, and cobalt.

[0031] For example, when sodium is used as an inorganic salt, NaCl, NaNO3, NaHCO3, Na2CO3, etc. may be used.

[0032] It is preferable to use compounds that serve as nitrogen and phosphorus sources for the halophilic bacteria as these inorganic salts.

[0033] The nitrogen source is not particularly limited and may be nitrate, nitrite, urea, ammonium salt, glutamate, or the like, and may be, for example, NaNO3, NaNO2, NH4Cl, or other compounds.

[0034] The amount of nitrogen source used may be appropriately determined within a range that does not affect the growth of the bacterial cells and achieves the objective of optical resolution. Specifically, the amount of nitrate used is usually about 500 mg or more, more preferably about 1000 mg or more, and even more preferably about 1250 mg or more per 100 ml of medium at the initial stage of culture.

[0035] The phosphorus source may be a phosphate, monohydrogen phosphate, dihydrogen phosphate, or the like, and is not particularly limited, but may be, for example, a compound such as K2HPO4 or KH2PO4.

[0036] The amount of phosphorus source used may be determined appropriately from the same viewpoint as the amount of nitrogen source used. Specifically, the amount of dihydrogen phosphate used is usually about 50 to 400 mg, more preferably about 100 to 200 mg, per 100 ml of medium.

[0037] These inorganic salts may be used alone or in combination of two or more.

[0038] The medium used in the aerobic culture step contains only racemic 3HB or a salt thereof as an organic carbon source. Racemic 3HB is a mixture of (R)-3-hydroxybutyric acid and (S)-3-hydroxybutyric acid and is obtained, for example, by conventional organic synthesis, not asymmetric synthesis.

[0039] The concentration of racemic 3HB as an organic carbon source may be appropriately set within a range that allows for preferential uptake of R-3HB into the bacterial cells, promotes accumulation of PHB, and achieves the objective of optically resolving racemic 3HB.

[0040] In the aerobic culture step of the optical resolution method according to the present invention, halophilic bacteria belonging to the genus Halomonas are cultured in a medium having a relatively high salt concentration, so there is almost no risk of contamination or proliferation of other bacteria, etc. Therefore, the medium may or may not be sterilized, and the culture can be carried out using simple equipment.

[0041] The halophilic bacteria in the aerobic culture step are cultured under aerobic conditions. The conditions for the aerobic culture are not particularly limited, as long as the bacteria grow and R-3HB is taken up into the bacteria, which then accumulates as PHB.

[0042] Specifically, the halophilic bacteria are inoculated into approximately 5 ml of medium and pre-cultured overnight with shaking at a predetermined stirring speed and temperature. The bacterial cells obtained from the pre-culture are then diluted approximately 100-fold in medium contained in an Erlenmeyer flask, fermenter, jar fermenter, or the like, and main culture (corresponding to aerobic culture in this application) is carried out.

[0043] The culture temperature for the main culture can usually be set within a range of about 20 to 45°C, but is preferably set within a range of about 30 to 37°C. When an Erlenmeyer flask is used, the stirring speed can usually be set within a range of about 120 to 250 rpm, but is preferably set within a range of about 150 to 200 rpm. When a fermenter or jar fermenter is used, it is preferable to supply oxygen at an oxygen supply rate comparable to that described above.

[0044] In the aerobic culture step, halophilic bacteria belonging to the genus Halomonas may be aerobically cultured under these culture conditions. Specifically, the dissolved oxygen concentration in the medium during aerobic culture is not particularly limited, but is usually 0 mg / L or higher, and preferably 7 mg / L or higher.

[0045] The culture method in the aerobic culture step includes, but is not limited to, batch culture, semi-batch culture, continuous culture, and the like. However, considering that the halophilic bacteria used in the optical resolution method of the present invention are highly unlikely to be contaminated with other bacteria, long-term continuous culture is also possible. The culture environment may be one in which the medium is exposed to air, and may be adjusted by actively blowing an oxygen-containing gas onto the surface of the medium or by blowing such a gas into the medium. The culture environment may be either a non-sterile environment or a sterile environment.

[0046] The aerobic culture time is the time required for R-3HB to disappear from the medium and for S-3HB to remain, and is, for example, 20 to 50 hours. Preferably, the presence of R-3HB in the medium is continuously confirmed using an analytical method such as HPLC, and once R-3HB has disappeared, the separation step described below is carried out and the culture is stopped.

[0047] [Separation process] The separation step in the optical resolution method of the present invention is a step of separating the bacterial cells from the culture medium after the aerobic culture step, and the aerobic culture step is terminated by separating the bacterial cells from the culture medium.

[0048] In the separation step, the bacterial cells are separated from the culture medium using a known method, and the aerobic culture step is then terminated. For example, when a liquid medium is used for the aerobic culture step, the culture solution obtained in this step contains S-3HB or a salt thereof that has not been taken up by the halophilic bacteria. Therefore, by separating the halophilic bacteria from the culture solution using a separation means and then terminating the aerobic culture step, a culture solution containing S-3HB or a salt thereof but containing no or almost no R-3HB can be obtained.

[0049] As a specific separation method, known solid-liquid separation procedures such as centrifugation and filtration can be used.

[0050] [Microaerobic culture process] The microaerophilic culturing step in the optical resolution method of the present invention is a step of culturing the bacterial cells separated in the separation step in a medium under microaerophilic conditions to produce (R)-3-hydroxybutyric acid or a salt thereof in the culture medium.

[0051] The phrase "producing (R)-3-hydroxybutyric acid in the culture medium" refers to the secretion of 3HB by the halophilic bacteria into the culture medium. The phrase "producing a salt of (R)-3-hydroxybutyric acid in the culture medium" refers not only to the secretion of a 3HB salt by the halophilic bacteria into the culture medium, but also to the reaction of the 3HB secreted by the halophilic bacteria with cationic components present in the culture medium to form an R-3HB salt. The cationic components that react with R-3HB to form a salt are not particularly limited as long as they are present in the culture medium, but include, for example, sodium ions, potassium ions, calcium ions, magnesium ions, cobalt ions, zinc ions, iron ions, copper ions, molybdenum ions, ammonium ions, and manganese ions.

[0052] Specifically, this is a process in which halophilic bacteria are cultured by adjusting and / or maintaining the pH in the alkaline range of 8.0 or higher, and R-3HB or a salt thereof is produced under conditions in which oxygen is not actively supplied to the halophilic bacteria in the culture medium. If the culture is continued without actively supplying oxygen to the halophilic bacteria, the oxygen in the system is consumed and the system becomes nearly anaerobic. However, an environment with an oxygen concentration of several percent is maintained, which does not reach absolute anaerobic state, and therefore an environment suitable for microaerobic culture can be maintained.

[0053] The medium used in the microaerobic culture step contains inorganic salts but does not contain an organic carbon source, and the same medium as that used in the aerobic culture step can be used except that it does not contain an organic carbon source.

[0054] The conditions for microaerobic culture are not particularly limited as long as they allow PHB accumulated within the bacterial cells to be secreted into the medium as R-3HB.

[0055] When microaerobic cultivation is continued, the pH of the medium tends to decrease due to the production of organic acids. The pH of such a medium can be appropriately confirmed using a known pH measuring device or a jar fermenter equipped with such a device.

[0056] The term "adjusting and / or maintaining" means maintaining a suitable pH state by adding a pH adjuster while checking the pH as described above, or simply adding a pH adjuster to adjust the pH at the start of culture, without subsequently adjusting the pH.

[0057] The pH adjusted and maintained in the microaerobic culture step is preferably 8.0 or higher, which allows the productivity of R-3HB by halophilic bacteria to be maintained at a high level.

[0058] The halophilic bacteria used in the present invention can be cultured under moderately high salt concentrations and alkaline conditions, which reduces the risk of contamination with foreign bacteria. However, some lactic acid bacteria can grow in environments with moderately high salt concentrations and a pH of 8.4 or less. If such bacteria contaminate the culture system of the present invention, they may consume the R-3HB or a salt thereof secreted by the halophilic bacteria as a substrate for lactic acid fermentation, further decreasing the pH of the medium.

[0059] Therefore, in the present invention, in order to culture halophilic bacteria without sterilizing the medium and / or in a non-sterile environment and secrete 3HB into the medium, it is preferable to adjust and maintain the pH of the medium at about 8.5 or higher during the microaerobic culture process.

[0060] The pH adjuster is not particularly limited, but examples thereof include hydroxides, carbonates, bicarbonates, etc. More specific examples include sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, sodium carbonate, potassium carbonate, calcium carbonate, magnesium carbonate, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium bicarbonate, and aqueous ammonia.

[0061] [R-isomer recovery process] The R-isomer recovery step in the optical resolution method of the present invention is a step of recovering (R)-3-hydroxybutyric acid or a salt thereof from the culture medium obtained in the microaerobic culture step.

[0062] In the R-isomer recovery step, R-3HB or a salt thereof can be recovered using known techniques. For example, when R-3HB or a salt thereof is present in the culture medium, the microaerobic culture step can be stopped, the culture medium containing R-3HB or a salt thereof can be separated from the bacterial cells, and R-3HB or a salt thereof can be recovered from the culture medium. Furthermore, when the microaerobic culture step is performed using a liquid medium, the culture medium obtained in this step contains secreted R-3HB or a salt thereof. Therefore, the culture in this step can be stopped, and if necessary, the culture medium can be separated from the halophilic bacteria using a separation means to obtain a culture medium, from which R-3HB or a salt thereof can be recovered.

[0063] Specific separation techniques that can be used include known solid-liquid separation procedures such as centrifugation and filtration. The method for terminating the culture is also not particularly limited. For example, a method of sterilizing the culture solution containing the halophilic bacteria by heating, acid treatment, or the like after the microaerobic culture step, or a method of separating the culture solution from the halophilic bacteria by solid-liquid separation, can be used.

[0064] The method for confirming the presence of R-3HB or a salt thereof in the culture medium varies depending on the bacterial species, medium components, culture conditions, etc., and should be appropriately determined taking these factors into consideration. For example, the culture medium can be sampled periodically and subjected to analytical methods such as HPLC to determine the time to stop the culture.

[0065] When R-3HB is present in the culture medium as an alkali metal salt formed by reaction with alkali metal or alkaline earth metal cations, such as sodium or calcium, based on inorganic salts contained in the culture medium, R-3HB can be obtained by subjecting the culture medium from which the bacterial cells have been separated to a conventional method, such as crystallization.

[0066] The culture medium after bacterial cell separation may be purified by column chromatography using an appropriate column to recover R-3HB. Alternatively, the pH of the culture medium after bacterial cell separation may be appropriately adjusted to purify the desired R-3HB or a salt thereof. Alternatively, the culture medium after bacterial cell separation may be added with a lower alcohol, which may be subjected to an esterification reaction, followed by purification as an R-3HB ester by distillation or other methods.

[0067] [S-body recovery process] The S-isomer recovery step in the optical resolution method of the present invention is a step of recovering (S)-3-hydroxybutyric acid or a salt thereof from the culture medium from which the bacterial cells have been separated in the separation step.

[0068] As described above, the culture medium from which the bacterial cells were separated in the separation step contains S-3HB or a salt thereof, but contains almost no R-3HB. In the S-isomer recovery step, (S)-3-hydroxybutyric acid (S-3HB) or a salt thereof can be recovered from the culture medium from which the bacterial cells were separated in this separation step using known techniques.

[0069] When S-3HB is present in the culture medium as an alkali metal salt formed by reaction with alkali metal or alkaline earth metal cations, such as sodium or calcium, based on inorganic salts contained in the culture medium, S-3HB can be obtained by subjecting the culture medium from which the bacterial cells have been separated to a conventional method, such as crystallization.

[0070] The culture medium after bacterial cell separation may be purified by column chromatography using an appropriate column to recover S-3HB. Alternatively, the pH of the culture medium after bacterial cell separation may be appropriately adjusted to purify the desired S-3HB or a salt thereof. Alternatively, the culture medium after bacterial cell separation may be added with a lower alcohol, which may be subjected to an esterification reaction, followed by purification as an S-3HB ester by distillation or other methods.

[0071] The present invention will be described in more detail below with reference to examples, although it goes without saying that the present invention is not limited to these examples.

[0072] As an example, racemic 3HB was optically resolved by the optical resolution method according to the present invention using Halomonas sp. KM-1 strain as a halophilic bacterium. Figure 1 shows an outline of the optical resolution method carried out as an example.

[0073] Specifically, the KM-1 strain was added to a baffled flask containing SOT medium with the composition shown in Table 1, which contained racemic 3-hydroxybutyrate (50% R-3HB and 50% S-3HB) as a carbon source, and aerobic culture was performed while shaking at a speed of 200 rpm or higher. The concentrations of R-3HB and S-3HB in the culture medium and the amount of PHB accumulated in the bacterial cells were measured over time using known methods. When R-3HB disappeared from the culture medium (31 hours after the start of culture as shown in Figure 2), the bacterial cells were separated from the culture medium and the aerobic culture was stopped.

[0074] [Table 1]

[0075] Figure 2 is a graph summarizing the analytical data from aerobic cultivation. As can be seen from the graph, the R-3HB concentration gradually decreased after the start of cultivation, reaching almost zero after 31 hours, while the S-3HB concentration did not show any significant decrease. Furthermore, the amount of PHB accumulated gradually increased inversely proportional to the decrease in R-3HB concentration. This clearly indicates that during the aerobic cultivation process, R-3HB is preferentially taken up by the KM-1 strain, which then accumulates as PHB within the bacterial cells.

[0076] Next, the bacterial cells separated from the culture medium were subjected to a microaerobic culture step using SOT medium (a medium with the composition shown in Table 1 excluding (R)-3-hydroxybutyric acid and (S)-3-hydroxybutyric acid) under conditions where oxygen was not actively supplied. After confirming that the amount of PHB accumulated in the bacterial cells was almost constant, the bacterial cells were separated from the culture medium 39 hours after the start of the culture, and the microaerobic culture was stopped.

[0077] Figure 3 is a graph summarizing the analytical data from microaerobic cultivation. As can be seen from the graph, the R-3HB concentration gradually increased for the first 15 hours after the start of cultivation and then remained nearly constant, while the S-3HB concentration remained unchanged at nearly zero. Furthermore, the amount of PHB accumulated gradually decreased inversely proportional to the increase in R-3HB concentration and remained nearly constant after 15 hours after the start of cultivation. This clearly demonstrates that during the microaerobic cultivation process, PHB accumulated within the KM-1 strain is secreted and produced as R-3HB into the culture medium.

[0078] Furthermore, S-3HB with an optical purity of 95% or more could be recovered from the culture medium from which the bacterial cells were separated after the aerobic culture step, and R-3HB with an optical purity of 95% or more could be recovered from the culture medium from which the bacterial cells were separated after the microaerobic culture step. Therefore, it was confirmed that the optical resolution method of the present invention can produce optically active 3-hydroxybutyric acid. [Industrial Applicability]

[0079] The present invention can be applied to a method for optically resolving racemic 3-hydroxybutyric acid.

Claims

1. A method for optical resolution of racemic 3-hydroxybutyric acid or a salt thereof, comprising the steps of: an aerobic culturing step of aerobically culturing a halophilic bacterium belonging to the genus Halomonas in a medium containing inorganic salts and racemic 3-hydroxybutyric acid or a salt thereof as an organic carbon source; a separation step of separating bacterial cells from the culture solution after the aerobic culture step; a microaerophilic culturing step in which the bacterial cells separated in the separation step are microaerophilically cultured in a medium containing inorganic salts to produce (R)-3-hydroxybutyric acid or a salt thereof in the culture solution, in this method for optical resolution of racemic 3-hydroxybutyric acid,

2. 2. The method for optical resolution of racemic 3-hydroxybutyric acid according to claim 1, further comprising an R-isomer recovery step of recovering (R)-3-hydroxybutyric acid or a salt thereof from the culture medium obtained in the microaerobic culture step.

3. 3. The method for optical resolution of racemic 3-hydroxybutyric acid according to claim 1, further comprising an S-isomer recovery step of recovering (S)-3-hydroxybutyric acid or a salt thereof from the culture medium from which the bacterial cells have been separated in the separation step.

4. 4. The method for optical resolution of racemic 3-hydroxybutyric acid according to claim 1, wherein the halophilic bacterium is Halomonas sp. KM-1 strain.

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